PROJECT SUMMARY/ABSTRACT Cancer research has recently experienced a paradigm shift from the seemingly obvious target of tumor cells towards key support systems of cancer, such as the tumor microenvironment (TME). Tissue oxygenation (pO2) and acidosis (pH) are among the most established hallmarks in solid tumor. Recently, extracellular inorganic phosphate (Pi) has been identified as a new signaling molecule of importance in tumorigenesis. This project aims to develop new biocompatible paramagnetic probes suitable for systemic delivery allowing for in vivo concurrent measurement of tissue acidosis (pH), oxygenation (pO2) and Pi concentration in the extracellular compartment (HOPE probes) using electron paramagnetic resonance (EPR)-based technologies. These developments will provide a new unique tool in cancer research allowing for in vivo direct non-invasive measurements and correlation of these important TME parameters. Under SA1, a robust click chemistry approach has been designed to efficiently produce a library of PEGylated HOPE probes. Under SA2, tumor targeting will be achieved upon conjugation of the PEGylated probe to RGD and/or folic acid ligands. The synthesized probes will be evaluated in vitro for their functional sensitivity, spectral properties, toxicity and ability to bind to the targeted biological receptors. Finally, under SA3, we will use a mouse model of human breast cancer to perform toxicokinetic studies, to optimize the probe dose and experimental time window, and to determine the functional sensitivity and tumor targeting efficiency of the probes using low frequency EPR spectroscopy and Overhauser-enhanced magnetic resonance imaging. We anticipate to achieve in vivo functional sensitivity of 1-2 mmHg of pO2, 0.05 units of pH, and 0.1 mM of Pi. We expect the dual targeted approach of the two receptors upregulated in numerous cancer types to improve the contrast between healthy and tumor tissues, and to significantly enhance signal intensity and decrease probe dosage. The completion of this K99/R00 award by the PI will allow him to obtain the necessary tools and skills in EPR-based in vivo spectroscopy and imaging to successfully apply this knowledge to the field of cancer functional imaging; therefore, bridging the interface between synthetic organic chemistry, cancer research and imaging technologies. The successful completion of this project is designed to make a signficant impact on the future of bioimaging applications to medicine.